Diabetes is a major public health problem. In the United States, sixteen million persons have diabetes (American Diabetes Association, Professional Section Quarterly, Summer 1998). Ocular complications of diabetes are the leading cause of new cases of legal blindness in people ages 20 to 74 in the United States. The risk for lower extremity amputation is 15 times greater in individuals with diabetes than in individuals without it. Kidney disease is a frequent and serious complication of diabetes. Approximately 30 percent of all new patients in the United States being treated for end-stage renal disease have diabetes. Individuals with diabetes are also at increased risk for periodontal disease. Periodontal infections advance rapidly and lead not only to loss of teeth but also to compromised metabolic function. Women with diabetes risk serious complications of pregnancy. Current statistics suggest that the mortality rates for infants of mothers with diabetes is approximately 7 percent.
Clearly, the economic burden of diabetes is enormous. Each year, patients with diabetes or its complications spend 24 million patient-days in hospitals. Diabetes is our nation's most expensive disease with an estimated total annual cost of $98 billion; however, the full economic impact of this disease is even greater because additional medical expenses often are attributed to the specific complications of diabetes rather than to diabetes itself.
Diabetes is a chronic, complex metabolic disease that results in the inability of the body to properly maintain and use carbohydrates, fats, and proteins. It results from the interaction of various hereditary and environmental factors and is characterized by high blood glucose levels caused by a deficiency in insulin production or an impairment of its utilization. Most cases of diabetes fall into two clinical types: Type I, or juvenile-onset, and Type II, or adult-onset. Type I diabetes is often referred to as Insulin Dependent Diabetes, or IDD. Each type has a different prognosis, treatment, and cause.
Approximately 5 to 10 percent of diabetes patients have IDD. IDD is characterized by a partial or complete inability to produce insulin usually due to destruction of the insulin-producing cells of the pancreatic islets of Langerhans. Patients with IDD would die without daily insulin injections to control their disease. Additionally, a fraction of Type II diabetics are insulin dependent and require insulin injections to improve their insulin resistance. Both Type I and insulin-dependent Type II diabetics can benefit from improvements in insulin administration, such as those described herein.
Few advancements in resolving the pathogenesis of diabetes were made until the mid-1970s when evidence began to accumulate to suggest that Type I IDD had an autoimmune etiopathogenesis. It is now generally accepted that IDD results from a progressive autoimmune response which selectively destroys the insulin-producing cells of the pancreatic Islets of Langerhans in individuals who are genetically predisposed. Autoimmunity to the cell in IDD involves both humoral (Baekkeskov et al. (1982) Nature 298:167; Baekkeskov et al. (1990) Nature 347:151; Reddy et al. (1988) Diabetologia 31:322; Pontesilli et al. (1987) Clin. Exp. Immunol. 70:84) and cell-mediated (Reddy et al. (1988); Pontesilli et al. (1987); Wang et al. (1987) Diabetes 36:535) immune mechanisms. Humoral immunity is characterized by the appearance of autoantibodies to cell membranes (anti-69 kD and islet-cell surface autoantibodies), cell contents (anti-carboxypeptidase A1, anti-64 kD and/or anti-GAD autoantibody), and/or cell secretory products (anti-insulin). While serum does not transfer IDD, anti-cell autoantibody occurs at a very early age, raising the question of an environmental trigger, possibly involving antigenic mimicry. The presence of cell-mediated immunological reactivity in the natural course of IDD is evidenced by an inflammatory lesion within the pancreatic islets, termed insulitis. Insulitis, in which inflammatory/immune cell infiltrates are clearly visible by histology, has been shown to be comprised of numerous cell types, including T and B lymphocytes, monocytes and natural killer cells (Signore et al. (1989) Diabetologia 32:282; and Jarpe et al. (1991) Regional Immunol. 3:305). Adoptive transfer experiments using the NOD (non-obese diabetic) mouse as a model of human IDD have firmly established a primary role for auto-aggressive T lymphocytes in the pathogenesis of IDD (Bendelac, et al. (1987) J. Exp. Med. 166:823; Miller et al. (1988) J. Immunol. 140:52; Hanafusa et al. (1988) Diabetes 37:204; and Bendelac et al. (1988) J. Immunol. 141:2625). Unfortunately, the mechanisms underlying destruction of the pancreatic cells remain unknown.
The mammalian pancreas controls nutrient resorption and glucose metabolism through its major components, the ductal cells, acinar cells and endocrine cells. The endocrine cells include insulin-produce β cells. Despite the fact that all three components of the pancreas differ in functionality, they are all of the same origin, the primitive gut endoderm. During early gestation (28 days in humans) evaginations of the embryonal foregut form the ventral and dorsal buds of the pancreas. The two buds arise opposite to each other while the gut is still surrounded by primitive mesenchyme. After rotation of the stomach and duodenum, the ventral anlage moves around and fuses with the dorsal bud. The ventral bud forms the posterior part of the pancreatic head including the ulcinate process, while the dorsal bud forms the remainder of the organ. In the enlarging epithelial buds, a treelike ductal system develops which eventually gives rise to endocrine and acinar cells (Peters et al. (2000) Virchows Arch. 436:527–538). It is believed that the “protodifferentiated” epithelial cells which reside in the ducts also share the features of ductal cells (Pictet et al. (1972) Development of the embryonic endocrine pancreas, In: Geiger S R (ed.) Handbook of Physiology, sect 7: Endocrinology, Waverley Press, Baltimore, pp25–66). These and more recent observations suggest that the pancreatic duct cells harbor the stem cells, which under appropriate stimuli, can give rise to acinar and endocrine cells (Ramiya V. et al. (2000) Nat. Med. 6:278–282; Bonner-Weir et al. (2000) Proc. Natl. Acad. Sci. USA 97:7999–8004). The pancreatic ductal progenitor stem cells have been shown to express tyrosine hydroxylase (Teitelman et al. (1993) Development 118:1031–1039), glucose transporter (GLUT-2) (Pang et al. (1994) Proc. Natl. Acad. Sci. USA 91:9559–9563), cytokeratins (Bouwens et al. (1994) Diabetes 43:1279–1283), Pdx-1 (Jonsson et al. (1994) Nature 371:606–609), high-affinity nerve growth factor TrkA (Kanaka-Gantenbein et al. (1995) Endocrinology 136:3154–3162), Isl-1 (Ahlgren et al. (1997) Nature 385:257–60), and ngn-3 (Gradwohl et al. (2000) Proc. Natl. Acad. Sci. USA 97:1607–1611). In the human fetal pancreas, proliferation is mainly found in the ductal cell compartment, followed in frequency by endocrine cells, which are synaptophysin positive but hormone negative, and finally, insulin or glucagon positive cells. In addition, it was noted that all epithelial cells, including endocrine cells, express cytokeratin 19 from 12–16 gestation weeks. The cytokeratin disappears later from the endocrine cells (Bouwens et al. (1997) Diabetologia 40:398–404).
For IDD patients, regular insulin injections do not maintain blood glucose near normal levels at all times and consequently patients develop secondary complications. While pancreatic and islet transplantations can consistently establish a euglycemic state and significantly reduce long-term complications, availability of the grafts is severely limited. Xenotransplants, on the other hand, pose a potentially serious threat of xenosis (transfer of animal infections to humans) with attendant regulatory problems and delays. Thus, there is an urgency to develop a pancreatic endocrine replacement therapy for Type 1 diabetic patients that would supply a sufficient number of functional human islets or their equivalents on demand.
One response to this need has been to develop in vitro culture methods for pancreatic differentiated cells or tumor cells (e.g., Gazdar et al. (1980) Proc. Natl. Acad. Sci. 77(6):3519–3525; Brothers, A., WO 93/00441; Korsgren et al. (1993) J. Med. Sci. 98(1):39–52; Nielson, J., WO 86/01530; McEvoy et al. (1982) Endocrinol. 111(5):1568–1575; Zayas et al., EP 0 363 125; and Coon et al., WO 94/23572). Such culture methods could be used to generate endocrine hormones or, in some instances, furnish cells for transplantation.
Another response has been to identify and culture pancreatic stem cells which can give rise to islet progenitor cells (IPCs) and IPC-derived islets (IdIs) or islet-like structures (see U.S. Ser. No. 09/406,253, filed Sep. 27, 1999, and Peck et al., WO 01/23528). The advantages of this method include the long-term propagation of the stem cells and the use of the stem cells and their progeny for implantation into patients, wherein they proliferate to form a pancreas-like structure that can restore euglycemia.
Notwithstanding the foregoing, there remains a need to develop additional pancreatic endocrine replacement therapies. The subject invention concerns the use of non-pancreatic stem cells in the development of therapies for IDD. Specifically, non-pancreatic stem cells are transdifferentiated to the pancreatic lineage.
A stem cell is a cell that has the capacity to both self-renew and to generate differentiated progeny. Two stem cells that are already in clinical use are hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). The mammalian hematopoietic system produces at least eight distinct lineages of mature blood cells in a continuous manner throughout adult life. These lineages include red blood cells, monocytic, granulocytic, basophilic, myeloid cells, the T and B cells and platelets. In this sense, hematopoiesis may resemble other developmental systems such as small intestine, epidermis, and hair follicle of the skin as well as the male germ cells. Other tissue types such as the liver, central nervous system and skeletal muscles seem to replenish more slowly, or in response to injury (Lemischka et al. (1986) Cell 45:917–927). Complex quantitative analyses of HSCs, in some cases, demonstrated that a single transplantable stem cell is both necessary and sufficient to transfer an intact, normal hematopoietic system to a recipient host (Jordan et al. (1990) Genes Dev. 4:220–232; and Smith L. et al. (1991) Proc. Natl. Acad. Sci. 88:2788–2792).
The proliferation and development of HSCs in vivo is promoted by contact with bone marrow stromal cells and the surrounding extracellular matrix. While there is some ability of soluble cytokines or growth factors to promote survival and proliferation of stem cells and their progeny in the absence of stromal cell matrix, the primitive HSCs can only be maintained, in the long term, when co-cultured with the appropriate stromal cell environment (Dexter et al. (1990) Ciba Found. Symp. 148:76–86). The characterization of CD34 antigen on HSCs, expressed only by 0.5–5% of human bone marrow cells, has enabled the purification of HSCs in commercial quantities. CD34 is not expressed on more mature counterparts (Civin et al. (1990) Prog. Clin. Biol. Res. 333:387–401). Using the long term bone marrow culture system, it has been established that CD34+ HSCs can survive in vitro and differentiate when allowed to grow in contact with bone marrow derived stromal cells, which produce a plethora of factors including M-CSF, GM-CSF, G-CSF, IL-1, IL-6, IL-7, TGF-beta, LIF, SCF (Heyworth et al. (1997) In: Stem Cells, Academic Press Ltd., pp243–441).
Both HSCs and MHCs have been suggested to share common bone marrow precursors that express CD34 antigen. Accordingly, CD50− and CD34+ cells give rise to MSCs, while CD50+ CD34+ cells give rise to HSCs. Also, circulating cells include fibroblast-like MSCs (also called fibrocytes) along with HSCs. The MSCs can differentiated into osteocytes, adipocytes and chondrocytes in vitro when appropriate growth factors are provided (Pittenger et al. (1999) Science 284:143–146).
Although less extensive, other studies have identified candidate stem cells from a number of other tissues (Reynolds et al. (1992) Science 255:1707–1710; Johansson et al. (1999) Cell 96:25–34; Potten et al., Int. J. Exp. Pathol. 78:219–243; Watt F (1998) Philos. Trans. R. Soc. London B 353:831–837; Alison M (1998) Curr. Opin. Cell Biol. 10:710–715; and Ramiya V. et al. (2000) Nat. Med. 6:278–282).
Recent progress in stem cell biology puts the traditional view that a cell's fate is sealed when it becomes part of endoderm, mesoderm, or ectoderm, the primary germ layers of the embryo, in paradox. More specifically, the assumption that the undifferentiated stem cell state as defined by the ability of stem cell to produce mature cell populations is limited to the range of cell types characteristic of each individual tissue (by implication, any given somatic stem cell is physically resident within its appropriate tissue) has been called into question (Lemischka I. (1999) Proc. Natl. Acad. Sci. USA 96:14193–14195). For instance, after transplantation into irradiated hosts, genetically labeled neural stem cells were found to produce a variety of blood cell types including myeloid and lymphoid cells as well as early hematopoietic cells (Bjornson et al. (1999) Science 283:534–537). The muscle tissue has been shown to contain a population of stem cells with several characteristics of bone marrow-derived HSCs, including high efflux of the fluourescent dye Hoechst 33342 and expression of the stem cell antigens Sca-1 and c-Kit but not CD45. These stem cells have been suggested to be identical to muscle satellite cells, some of which lack myogenic regulators and which are capable of responding to hematopoietic signals (Jackson et al. (1999) Proc. Natl. Acad. Sci. USA 96:14482–14486). A similar observation has been made by another group who also demonstrated the muscle differentiation potential of a subset of bone marrow-derived stem cells (Gussoni et al. (1999) Nature 401:390–394). A possibility of HSCs mobilizing during liver failure to increase the regenerative capacity of the liver, though to a lesser extent, has been documented (Alison et al. (2000) Nature 406: 257). These observations invite speculation that the functional plasticity of somatic tissue derived stem cells may be greater than expected.
HSCs and MSCs are presently routinely used in the clinical setting and are commercially available. They can be purified from the peripheral blood or bone marrow using commercially available technology. Obtaining pancreatic endocrine tissue or hormones from HSCs or MSCs that have committed to the pancreatic differentiation pathway would be very useful in advancing the treatment of diabetes.